The cornerstones of prophylaxis and treatment of infections caused by non-tuberculosis mycobacteria (NTM) are the macrolides, clarithromycin and azithromycin. Although clinically acquired macrolide resistance in mycobacteria is conferred by 23S rRNA gene mutation, intrinsic resistance involves expression of adenine rRNA methylases, or erm genes. Moreover, recent data indicates that other genes can significantly affect the antimycobacterial activity of macrolides. For instance, an rpsA gene allele confers high-level macrolide resistance, and evidence suggests that disruption of trans-translation increases the bactericidal activity of macrolides. In addition, Mycobacteria may have a macrolide efflux system. Therefore, we hypothesize that mycobacteria may have several mechanisms that reduce the antimicrobial effects of macrolides. These mechanisms may include target modification (e.g., rRNA methylation or mutation), target recovery (e.g., trans-translation), and drug transport (e.g., efflux). Consequently, the long-term objective of this work is to characterize the mechanisms that affect the susceptibility of mycobacteria to macrolides. To address this objective, the project is divided into 4 specific aims: (1) to characterize the erm genes of mycobacteria;(2) to correlate the function of the rpsA and smpB genes with resistance to and recovery from the affects of macrolides;(3) to characterize the putative membrane protein genes that affect susceptibility to macrolides;and (4) to define the distribution of genes that affect the activity of macrolides within the Mycobacteriaceae. Mycobacteria are important human pathogens that cause chronic, often intractable disease. Understanding resistance and other processes that reduce drug activity will aid the design and development of new agents with activity against mycobacteria and/or treatment regimens.